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Method to increase the yield of eukaryotic membrane protein expression in Saccharomyces cerevisiae for structural and functional studies.

Parker JL, Newstead S - Protein Sci. (2014)

Bottom Line: The production of milligram quantities of recombinant protein is still a serious obstacle to the structural and functional characterization of these proteins.We further demonstrate that the increase in expression for our test proteins resulted in a concomitant increase in functional protein.Using this system, we were able to increase the expression level of a plant transporter, NRT1.1, which was a key factor in its structural and functional characterization.

View Article: PubMed Central - PubMed

Affiliation: Department of Biochemistry, University of Oxford, South Parks Road, Oxford, OX1 3QU, United Kingdom.

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Analysis of the quality of the protein. (A) Representative FSEC analysis of three of the constructs obtained under both—uracil and—leucine media. (B) Gel filtration profile and SDS PAGE gel (inset) of the final step of purification of AtPTR1, please note that AtPTR1 runs as both a monomer and dimer on SDS PAGE but is monomeric by gel filtration. (C) Liposomes containing AtPTR1 can uptake tritiated di-alanine and this is proton dependent as the addition of the proton ionophore, CCCP, results in no uptake.
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fig02: Analysis of the quality of the protein. (A) Representative FSEC analysis of three of the constructs obtained under both—uracil and—leucine media. (B) Gel filtration profile and SDS PAGE gel (inset) of the final step of purification of AtPTR1, please note that AtPTR1 runs as both a monomer and dimer on SDS PAGE but is monomeric by gel filtration. (C) Liposomes containing AtPTR1 can uptake tritiated di-alanine and this is proton dependent as the addition of the proton ionophore, CCCP, results in no uptake.

Mentions: The newly constructed vector pDDGFP-Leu2d, then, allows for dual selection using either the URA3 or the LEU2D markers. Due to the shortening and, hence, weakening of the promoter for the LEU2 gene, for the yeast to grow under the selective pressure of minus leucine medium the pDDGFP-Leu2d plasmid has to be maintained at a high copy number; 80–100 copies per cell compared to approximately 20 for the URA3 marker. This increase in copy number can in turn lead to an increase in protein level.21 Nine different MPs, encompassing five different transporter families and four different eukaryotic organisms (both unicellular and multicellular) were screened (Table I) and in all cases a clear increase of between 2.9 and 5.7 times more fluorescence was measured from cells grown under minus leucine (−Leu) conditions compared to minus uracil (−Ura) for all proteins tested [Fig. 1(B)]. In addition, different yeast backgrounds were screened and in most cases again an increase in fluorescence was observed for −Leu conditions when compared to −Ura. The smallest difference was seen for the DF5 background where, under both selection conditions, only poor expression was seen [Fig. 1(C)]. To ensure the protein was of good quality and the increased expression level did not have a detrimental affect on the protein (i.e., aggregation or insoluble material), FSEC analysis was performed. In all cases, the constructs produced monodisperse traces, indicating that the increased expression did not lead to increased aggregation over expression in the –Ura medium [representative traces are shown in Fig. 2(A)]. Numerous targets have been produced and purified in milligram amounts from this system, for brevity here we show the purification [Fig. 2(B)] and functional characterization [Fig. 2(C)] of AtPTR1 a proton coupled peptide transporter from A. thaliana. Using this system, the final yield of purified, monodisperse, and functional AtPTR1 increased from 0.2 to 0.8 mg L−1.


Method to increase the yield of eukaryotic membrane protein expression in Saccharomyces cerevisiae for structural and functional studies.

Parker JL, Newstead S - Protein Sci. (2014)

Analysis of the quality of the protein. (A) Representative FSEC analysis of three of the constructs obtained under both—uracil and—leucine media. (B) Gel filtration profile and SDS PAGE gel (inset) of the final step of purification of AtPTR1, please note that AtPTR1 runs as both a monomer and dimer on SDS PAGE but is monomeric by gel filtration. (C) Liposomes containing AtPTR1 can uptake tritiated di-alanine and this is proton dependent as the addition of the proton ionophore, CCCP, results in no uptake.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4230410&req=5

fig02: Analysis of the quality of the protein. (A) Representative FSEC analysis of three of the constructs obtained under both—uracil and—leucine media. (B) Gel filtration profile and SDS PAGE gel (inset) of the final step of purification of AtPTR1, please note that AtPTR1 runs as both a monomer and dimer on SDS PAGE but is monomeric by gel filtration. (C) Liposomes containing AtPTR1 can uptake tritiated di-alanine and this is proton dependent as the addition of the proton ionophore, CCCP, results in no uptake.
Mentions: The newly constructed vector pDDGFP-Leu2d, then, allows for dual selection using either the URA3 or the LEU2D markers. Due to the shortening and, hence, weakening of the promoter for the LEU2 gene, for the yeast to grow under the selective pressure of minus leucine medium the pDDGFP-Leu2d plasmid has to be maintained at a high copy number; 80–100 copies per cell compared to approximately 20 for the URA3 marker. This increase in copy number can in turn lead to an increase in protein level.21 Nine different MPs, encompassing five different transporter families and four different eukaryotic organisms (both unicellular and multicellular) were screened (Table I) and in all cases a clear increase of between 2.9 and 5.7 times more fluorescence was measured from cells grown under minus leucine (−Leu) conditions compared to minus uracil (−Ura) for all proteins tested [Fig. 1(B)]. In addition, different yeast backgrounds were screened and in most cases again an increase in fluorescence was observed for −Leu conditions when compared to −Ura. The smallest difference was seen for the DF5 background where, under both selection conditions, only poor expression was seen [Fig. 1(C)]. To ensure the protein was of good quality and the increased expression level did not have a detrimental affect on the protein (i.e., aggregation or insoluble material), FSEC analysis was performed. In all cases, the constructs produced monodisperse traces, indicating that the increased expression did not lead to increased aggregation over expression in the –Ura medium [representative traces are shown in Fig. 2(A)]. Numerous targets have been produced and purified in milligram amounts from this system, for brevity here we show the purification [Fig. 2(B)] and functional characterization [Fig. 2(C)] of AtPTR1 a proton coupled peptide transporter from A. thaliana. Using this system, the final yield of purified, monodisperse, and functional AtPTR1 increased from 0.2 to 0.8 mg L−1.

Bottom Line: The production of milligram quantities of recombinant protein is still a serious obstacle to the structural and functional characterization of these proteins.We further demonstrate that the increase in expression for our test proteins resulted in a concomitant increase in functional protein.Using this system, we were able to increase the expression level of a plant transporter, NRT1.1, which was a key factor in its structural and functional characterization.

View Article: PubMed Central - PubMed

Affiliation: Department of Biochemistry, University of Oxford, South Parks Road, Oxford, OX1 3QU, United Kingdom.

Show MeSH